Insulin resistance in skeletal muscle is a condition that is common to obesity, type 2 diabetes, and cardiovascular disease. These diseases affect millions of individuals worldwide. The insulin signaling pathway is crucial to a wide variety of biological processes in skeletal muscle such as glycogen synthesis and glucose transport. It involves precise, controlled protein-protein interactions as well as protein phosphorylation to relay the insulin signal. Defects in the insulin signaling pathway have been implicated in the development of skeletal muscle insulin resistance but the precise abnormalities in protein-protein interactions and protein phosphorylation are largely unclear. The present project will analyze proteins isolated from muscle biopsies of lean healthy, obese nondiabetic and type 2 diabetic volunteers utilizing state-of-the-art HPLC-nanospray- tandem mass spectrometry (HPLC-ESI-MS/MS) to assess interacting partners of insulin receptor substrate-1 (IRS-1) and to discover and quantify novel phosphorylation of proteins in the insulin signaling pathway. The central hypothesis of this investigation is that there are differences in protein-protein interactions and protein phosphorylation in the insulin signaling pathway in human skeletal muscle in obesity and type 2 diabetes as compared to lean and healthy conditions. The overall goal of our research is to identify molecular mechanisms responsible for insulin resistance in human skeletal muscle and to provide novel targets for prevention and treatment of type 2 diabetes. The outcome of this proposed work will be the discovery of novel protein complexes and phosphorylation sites associated with insulin resistance and regulated by insulin that will expedite the generation of hypotheses to tackle the challenging issues of type 2 diabetes. Specifically, we propose to: 1. Determine how protein-protein interactions involving IRS-1 in human skeletal muscle are altered in obesity and type 2 diabetes and are regulated by insulin. We will test the hypotheses that abnormal protein-protein complexes in human skeletal muscle are associated with insulin resistance that may impede signaling pathways involved in insulin action, and that these protein-protein interactions are regulated by insulin. We will perform co-immunoprecipitation experiments using a specific antibody against IRS-1, a major player in insulin signaling, followed by identification and quantification of IRS-1 associated proteins by HPLC-ESI-MS/MS. 2. Determine how phosphorylation of proteins involved in the insulin signaling pathway in skeletal muscle are dysfunctional in obesity and type 2 diabetes. We will test the hypothesis that abnormal phosphorylation patterns of proteins in the insulin signaling pathway in human skeletal muscle are associated with obesity and type 2 diabetes. We will employ the mass spectrometry based phosphorylation identification and quantification approach developed in our laboratory to target proteins of interest in the PI-3 kinase pathway, such as PI-3 kinase, PDK1, AKT and AS160. New and known phosphorylation sites will be quantified under both basal conditions and upon insulin infusion in vivo. .